TCR's are members of the immunoglobulin superfamily and usually consist of two subunits, namely the α- and β-subunits. These possess one N-terminal immunoglobulin (Ig)-variable (V) domain, one Ig-constant (C) domain, a transmembrane/cell membrane-spanning region, and a short cytoplasmic tail at the C-terminal end. The variable domains of both the TCR α-chain and β-chain have three hypervariable or complementarity determining regions (CDRs), whereas the variable region of the β-chain has an additional area of hypervariability (HV4) that does not normally contact antigen and therefore is not considered a CDR.
CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the β-chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC. CDR4 of the β-chain is not thought to participate in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which forms a link between the two chains.
The affinity of TCR's for a specific antigen makes them valuable for several therapeutic approaches. For example, cancer patients, such as melanoma patients, can be effectively treated by using adoptive immunotherapy.
The adoptive transfer of lymphocytes in the setting of allogeneic stem cell transplantation (SCT) has demonstrated the power of the immune system for eradicating hematological malignancies (Kolb et al. 1995). It appears that SCT can also function to eliminate solid tumors, such as renal cell carcinomas (RCC) in some cases (reviewed in Kolb et al. 2004 and Dudley and Rosenberg, 2003). In SCT recipients, the elimination of malignant cells may only occur after several months up to a year, due to the fact that specific T cells must be activated in vivo and must then expand to adequate numbers following the development of the new hematopoietic system in the transplant recipient. Alternatively, after a period of time (approximately 60 days) during which tolerance is established in the SCT recipient, a transfer of unprimed, unseparated lymphocytes can be made to speed up the generation of immune responses directed against tumor cells. Here again, the specific lymphocytes capable of attacking tumor cells must be activated and expanded from the low frequency precursor lymphocytes that are present among the unselected population of lymphocytes that are transferred. Donor lymphocyte infusions (DLI) of unselected lymphocyte populations after SCT work well for the elimination of chronic myelogenous leukemia (CML), which grows slowly, but are less effective in the eradication of acute leukemia, partly due to the fact that the growth of the malignant cells outpaces the expansion capacity of the immune cells. This same expansion differential in which immune cells expand more slowly than tumor cells, also impacts on the poor immune elimination of rapidly progressing solid tumors. A second handicap in the use of unselected mixed lymphocyte populations in DLI is that T cells may also be transferred that have the capacity to attack normal cells and tissues of the recipient, leading to graft-versus-host-disease (GVHD), a disease with high morbidity and mortality.
Recent studies have demonstrated that the adoptive transfer of selected T cells with defined peptide specificities can lead to major reductions in tumor burden in an autologous setting, particularly if patients have been pretreated with non-myeloablative regimens (Dudley et al. 2002, 2003). This eliminates the need to perform SCT in the tumor patient, and thereby also bypasses the problem of GVHD.
In order to extend the capacity to use adoptive cell therapy (ACT) to treat patients with more rapidly growing tumors, it is a goal to transfer enriched, peptide-specific effector T cells (both CD4 T helper cells and cytotoxic T lymphocytes) that have been selected for their ligand specificities to effectively attack tumor cells while avoiding serious attack of normal tissues. These cells are to be rapidly expanded to large numbers ex vivo and then used for ACT. Alternatively, the T cell receptors (TCR) of such ligand-specific T cells can be cloned and expressed as TCR-transgenes in activated lymphocytes, using either recipient peripheral blood lymphocytes or activated T cell clones with defined specificities that grow well and do not have the capacity to attack normal host tissues.
As examples, an expanded allospecific T cell clone that is specific for an MHC molecule not expressed by the recipient or an expanded T cell clone specific for a virus, such as cytomegalovirus or Epstein-Barr virus, could be used as recipient cells for the transgenic TCR. The availability of a panel of transgenic TCR vectors, recognizing different MHC-peptide ligands could be used to develop large numbers of pre-activated T cells of both the CD4 and CD8 subtypes, thereby allowing large numbers of effector lymphocytes to be rapidly prepared and transferred to patients whose tumors express the corresponding TCR ligands. This would save time in achieving the numbers of specific T cells required to control tumor growth, possibly leading to more effective tumor eradication of rapidly progressing tumors.
Because the determinants that specific T cells recognize on leukemia and lymphomas, as well as solid tumor cells, often represent self-peptides derived from over-expressed proteins that are presented by self-MHC molecules, the affinity of their T cell receptors (TCR) is low, since T cells bearing high affinity receptors have been eliminated through the process of negative selection which is applied to lymphocytes during their development in the thymus to prevent autoimmunity. More effective tumor cell recognition occurs if the T cells are generated from lymphocyte populations that have not been negatively selected against self-MHC-molecules during their development in the thymus.
WO 2006/031221 pertains to T cell receptors against tumor-associated antigens, nucleic acids encoding the same, vectors and cells comprising the nucleic acids encoding the T cell receptors, and methods of use thereof. Among others, it is disclosed that the TCR subunits have the ability to form TCR that confer specificity to T cells for tumor cells presenting MART-I, NY-ESO-I, and melanoma-related gp100.
In the prior art, several scientific and patent documents are existing, which describe TCR, which are able to recognise and bind tyrosinase. Visseren et al. (Int. J. Cancer (1997) 72, 1122-1128) describe the affinity and specificity of several tyrosinase-specific TCR and suggest to use these TCR as a specific treatment of melanoma patients.
Roszkowski et al. (J. Immunol. (2003) 170, 2582-2589 and Cancer Res. (2005) 65, 1570-1576) the like are characterising tyrosinase-specific TCR.
U.S. Pat. No. 5,906,936 is directed to cytotoxic T-cells which kill non-MHC-restricted target cells and not to cells, which comprise specific TCR sequences.
WO97/32603 is directed to a method for producing non-human TCR and TCR specific for human HLA-restricted tumor antigens. Furthermore, the TCR-nucleic acids and recombinant T-cells are described as well as the administration of TCR recombinant T-cells for the treatment of several diseases.
WO2007/065957 describes an effector T-cell transfected with an antigen specific TCR coding RNA wherein the transfected T-cell recognizes the antigen in a complex with the MHC-molecule and binds the same. As a potential tumor antigen, MART-1 (Melan-A), tyrosinase and survivin are named.
WO2008/039818 discloses MART-1 and tyrosinase-specific TCR sequences and describes the enhancement of antigen recognition by substitution in the CDR2 region.
The above prior art TCR sequences are all derived from autologous or xenogeneic, but not allogeneic, sources.
For example, TCR sequences are from peripheral blood or from tumor infiltrating lymphocytes of HLA-A2 positive melanoma patients. This means that all these TCR are HLA-A2 self-restricted TCRs, or, are HLA-DP4 restricted, NY-E50-1 specific, both derived from autologous sources. As an alternative, as disclosed in WO97/32603, the TCR is derived from an HLA-A2 transgenic mouse, so in this case the sequence is xenogeneic. However, the available prior art documents do not show TCR sequences, which are allo-restricted and tyrosinase-specific.
Thus, there is still an important need to find means to generate T cells that bear TCR with high functional avidity that have the capacity to recognize specific ligands on tumor cells. Although adoptive transfer of T cells expressing transgenic T cell receptors (TCR) with anti-tumor function is a hopeful new therapy for patients with advanced tumors, there is a critical bottleneck in identifying high-avidity T cells with TCR specificities needed to treat different malignancies.